METHODS

Heterologous cell model systems with stable expression of CRAF fusions were generated and used for testing downstream signaling pathways via immunoblotting. Soft agar assays and flank xenografts in immuno-compromised mice were used to characterize the oncogenic properties of CRAF fusions. Pathway activation and oncogenicity were further assessed in the presence of first – and second-generation RAF inhibitors, PLX4720 and PLX8394 respectively, MEKi, and mTOR inhibitors as single agents or in combination. Myc- and Flag-tagged constructs of CRAF fusions were used in co-immunoprecipitation assays to assess dimerization profiles of CRAF fusions with or without inhibitors.

RESULTS

CRAF fusions activated the MAPK and PI3K pathways. CRAF fusions can homo-dimerize as well as hetero-dimerize with full-length BRAF, CRAF and the N-terminal fusion partner protein. Compared to BRAF fusions, CRAF fusions were not found to be responsive to any RAF inhibitors tested, including the paradox breaker PLX8394. We found that while PLX8394 decreased BRAF fusion-mediated dimerization, dimerization of CRAF fusion is unaffected. Clinically relevant MEKi AZD6244 and GSK1120212 both suppressed CRAF fusion driven pathways and growth in vitro but in mice flank xenografts, GSK1120212 partially inhibited CRAF fusion driven tumors. Since the CRAF fusions also activate the PI3K pathway, combinatorial targeting using MEKi and mTOR inhibitor, GSK1120212 and RAD001 respectively, was found to show robust tumor inhibition in vivo.

CONCLUSIONS: This study demonstrates that CRAF fusions do not respond to RAF inhibitors, show partial response to single-agent MEKi, and respond robustly to combinatorial targeting of both MAPK and PI3K pathways via GSK1120212 and RAD001. Since PLX8394 does not affect CRAF fusion mediated dimerization, this provides a mechanistic basis for unresponsiveness to RAF inhibitors. Additionally, the N-terminal fusion partner also contributes to dimerization of CRAF fusions. Therefore, CRAF fusions are distinct from BRAF fusions in terms of responsiveness to targeted therapies due to dimerization profiles. These findings suggest molecular classification of PLGGs prior to treatment and provide preclinical rationale for combination therapy of CRAF fusion expressing PLGGs.

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